U.S. patent number 5,358,960 [Application Number 07/986,662] was granted by the patent office on 1994-10-25 for method for inhibiting advanced glycosylation of proteins using aminosubstituted imidazoles.
This patent grant is currently assigned to Alteon Inc., The Rockefeller University. Invention is credited to Anthony Cerami, Peter Ulrich, Dilip R. Wagle.
United States Patent |
5,358,960 |
Ulrich , et al. |
October 25, 1994 |
Method for inhibiting advanced glycosylation of proteins using
aminosubstituted imidazoles
Abstract
The present invention relates to compositions and methods for
inhibiting nonenzymatic cross-linking (protein aging), Accordingly,
a composition is disclosed which comprises 2-aminoimidazoles
capable of inhibiting the formation of advanced glycosylation
endproducts of target proteins by reacting with the carbonyl moiety
of the early glycosylation product of such target proteins formed
by their initial glycosylation, The method comprises contacting the
target protein with the composition, Both industrial and
therapeutic applications for the invention are envisioned, as food
spoilage and animal protein aging can be treated.
Inventors: |
Ulrich; Peter (Old Tappan,
NJ), Cerami; Anthony (Shelter Island, NY), Wagle; Dilip
R. (Valley Cottage, NY) |
Assignee: |
The Rockefeller University (New
York, NY)
Alteon Inc. (Northvale, NJ)
|
Family
ID: |
46202119 |
Appl.
No.: |
07/986,662 |
Filed: |
December 8, 1992 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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805200 |
Dec 10, 1991 |
5238963 |
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|
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481869 |
Feb 20, 1990 |
5128360 |
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220504 |
Jul 18, 1988 |
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798032 |
Nov 14, 1985 |
4758583 |
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590820 |
Mar 19, 1984 |
4665192 |
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Current U.S.
Class: |
514/400; 424/49;
514/398; 514/399; 514/866 |
Current CPC
Class: |
A61K
8/43 (20130101); A61K 31/155 (20130101); A61K
31/195 (20130101); A61K 31/415 (20130101); A61K
31/4178 (20130101); A61K 38/191 (20130101); A61K
38/2006 (20130101); A61K 38/217 (20130101); A61K
38/38 (20130101); A61K 39/0005 (20130101); A61Q
19/08 (20130101); C07D 405/14 (20130101); C07K
16/28 (20130101); G01N 33/533 (20130101); G01N
33/573 (20130101); G01N 33/582 (20130101); G01N
33/68 (20130101); G01N 33/6842 (20130101); G01N
33/6893 (20130101); A61K 31/155 (20130101); A61K
38/2006 (20130101); A61K 38/217 (20130101); A61K
38/38 (20130101); A61K 2039/6081 (20130101); G01N
2333/922 (20130101); A61K 2300/00 (20130101); A61K
2300/00 (20130101); A61K 2300/00 (20130101); A61K
2300/00 (20130101); Y10S 514/866 (20130101) |
Current International
Class: |
A61K
38/19 (20060101); A61K 39/00 (20060101); A61K
38/21 (20060101); A61K 38/38 (20060101); A61K
38/20 (20060101); A61K 31/185 (20060101); A61K
31/195 (20060101); A61K 31/155 (20060101); A61K
31/4164 (20060101); A61K 31/4178 (20060101); A61K
31/415 (20060101); A61Q 11/00 (20060101); C07D
405/14 (20060101); C07D 405/00 (20060101); C07K
16/18 (20060101); C07K 16/28 (20060101); G01N
33/573 (20060101); G01N 33/68 (20060101); G01N
33/58 (20060101); G01N 33/533 (20060101); A61K
031/415 () |
Field of
Search: |
;514/398,399,400 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
CA80:121003p, Chuiguk et al. (1974). .
Bucala et al., Advances in Pharmacology, 23:1-34, Academic Press
(1992). .
Eble et al., J. Biol. Chem., 258:9406-9412 (1983). .
Sell and Monnier, J. Biol. Chem. 264:21597-21602 (1989). .
Oimomi et al., Agric Biol. Chem. 53:(6):1767-1728 (1989). .
Oimomi et al., Diabetes Research and Clinical Practice, 6:311-313
(1989). .
Nicholas et al., Lab. Invest., 60 No. 4, p. 486 (1989). .
Brownlee et al., Science, 232:1629-1632 (1986). .
Brownlee et al., Diabetes, 35 Suppl. 1, p. 42A (1986). .
Nordbo, J. Dent. Res. 58:1429 (1979)..
|
Primary Examiner: Cintins; Marianne M.
Assistant Examiner: Jordan; Kimberly R.
Attorney, Agent or Firm: Klauber & Jackson
Parent Case Text
The present application is a continuation-in-part of co-pending
application Ser. No. 07/805,200 filed Dec. 10, 1991; and now U.S.
Pat. No. 5,238,963 which is a division of application Ser. No.
07/481,869, filed Feb. 20, 1990 and now U.S. Pat. No. 5,128,360;
which is a continuation-in-part of 07/220,504, filed Jul. 18, 1989,
now abandoned; which is a Division of U.S. Ser. No. 798,032, filed
Nov. 14, 1985 and now U.S. Pat. No. 4,758,583, which is a
continuation-in-part of U.S. Ser. No. 590,820, filed Mar. 19, 1984
and now U.S. Pat. No. 4,665,192. Applicants claim the benefits of
these applications under 35 U.S.C. 120.
Claims
What is claimed is:
1. A composition comprising an effective amount of a compound for
inhibiting the advanced glycosylation of a target protein, said
compound selected from the group consisting of compounds of the
formula ##STR3## wherein R.sup.1 is an amino group; R.sub.2 is
hydrogen, an amino group or a mono- or di-amino lower alkyl group;
R.sub.3 and R.sub.4 are independently hydrogen, a lower alkyl
group, an aryl group or an acyl group and their biologically or
pharmaceutically acceptable acid or alkali addition salts; and
mixtures thereof, and a pharmaceutical carrier thereof.
2. The composition of claim 1 wherein said compound has the formula
wherein R.sub.1 and R.sub.2 are both amino groups.
3. The composition of claim 2 wherein said compound is
1,2-diamino-4-phenyl-imidazole or a biologically acceptable salt
thereof.
4. The composition of claim 2 wherein said compound is
1,2-diaminoimidazole or a biologically acceptable salt thereof.
5. The composition of claim 1 wherein said compound has the formula
wherein R.sub.2 is a mono- or di-amino lower alkyl group.
6. The composition of claim 5 wherein said compound is
1-(2,3-diaminopropyl)imidazole or a biologically acceptable salt
thereof.
7. A pharmaceutical composition comprising a pharmaceutically
effective amount of a compound for administration to an animal to
inhibit the advanced glycosylation of a target protein within said
animal, said compound selected from the group consisting of
compounds of the formula ##STR4## wherein R.sub.1 is an a amino
group; R.sub.2 is hydrogen, an amino group or a mono- or di-amino
lower alkyl group; R.sub.3 and R.sub.4 are independently hydrogen,
a lower alkyl group, an aryl group or an acyl group and their
pharmaceutically acceptable acid or alkali addition salts; and
mixtures thereof, and a pharmaceutical carrier therefor.
8. The composition of claim 7 wherein said compound has the formula
wherein R.sub.1 and R.sub.2 are both amino groups.
9. The composition of claim 8 wherein said compound is
1,2-diamino-4-phenyl-imidazole or a pharmaceutically acceptable
salt thereof.
10. The composition of claim 7 wherein said compound is
1,2-diaminoimidazole or pharmaceutically acceptable salt
thereof.
11. The composition of claim 7 wherein said compound has the
formula wherein R.sub.2 is a mono-or diamino lower alkyl group.
12. The composition of claim 11 wherein said compound is
1-(2,3-diaminopropyl)imidazole or a pharmaceutically acceptable
salt thereof.
13. A method for inhibiting the advanced glycosylation of a target
protein comprising contacting the target protein with an effective
amount of composition comprising a compound selected from the group
consisting of compounds of the formula ##STR5## wherein R.sub.1 and
R.sub.2 are independently hydrogen, an amino group or a mono- or
di-amino lower alkyl group; R.sub.3 and R.sub.4 are independently
hydrogen, a lower alkyl group, an aryl group, or an acyl group with
the proviso that one of R.sub.1 and R.sub.2 must be an amino group
or an mono- or diamino lower alkyl group; and their biologically or
pharmaceutically acceptable acid or alkali addition salts; and
mixtures thereof, and a carrier therefor.
14. The method of claim 13 wherein said compound has the formula
wherein one of R.sub.1 and R.sub.2 an amino group.
15. The method of claim 14 wherein said compound is
2-aminoimidazole or a biologically acceptable salt thereof.
16. The method of claim 13 wherein said compound has the formula
wherein R.sub.1 and R.sub.2 are both amino groups.
17. The method of claim 16 wherein said compound is
1,2-diamino-4-phenyl-imidazole or a biologically acceptable salt
thereof.
18. The method of claim 16 wherein said compound is
1,2-diaminoimidazole or a biologically acceptable salt thereof.
19. The method of claim 13 wherein said compound has the formula
wherein one of R.sub.1 and R.sub.2 is a mono- or diamino lower
alkyl group.
20. The method of claim 19 wherein said compound is
1-(2,3-diaminopropyl)imidazole or a biologically acceptable salt
thereof.
21. A method for treating an animal to inhibit the formation of
advanced glycosylation endproducts of a target protein within said
animal, said method comprising administering an effective amount of
a pharmaceutical composition, said pharmaceutical composition
comprising a compound selected from the group consisting of
compounds of the formula ##STR6## wherein R.sub.1 and R.sub.2 are
independently hydrogen, an amino group or a mono- or di-amino lower
alkyl group; R.sub.3 and R.sub.4 are independently hydrogen, a
lower alkyl group, an aryl group, or an acyl group with the proviso
that one of R.sub.1 and R.sub.2 must be an amino group or an mono-
or diamino lower alkyl group; and their pharmaceutically acceptable
acid or alkali addition salts; and mixtures thereof, and a carrier
therefor.
22. The method of claim 21 wherein said compound has the formula
wherein one of R.sub.1 and R.sub.2 an amino group.
23. The method of claim 21 wherein said compound is
2-aminoimidazole or a pharmaceutically acceptable salt thereof.
24. The method of claim 21 wherein said compound has the formula
wherein R.sub.1 and R.sub.2 are both amino groups.
25. The method of claim 24 wherein said compound is
1,2-diamino-4-phenyl-imidazole or a pharmaceutically acceptable
salt thereof.
26. The method of claim 21 wherein said compound is
1,2-diaminoimidazole or a pharmaceutically acceptable salt
thereof.
27. The method of claim 21 wherein said compound has the formula
wherein one of R.sub.1 and R.sub.2 is a mono-or diamino lower alkyl
group.
28. The method of claim 27 wherein said compound is
1-(2,3-diaminopropyl)imidazole or a pharmaceutically acceptable
salt thereof.
29. A method of inhibiting the discoloration of teeth resulting
from non-enzymatic browning in the oral cavity which comprises
administration of an amount effective to inhibit the formation of
advanced glycosylation endproducts of a composition comprising a
compound selected from the group consisting of compounds of the
formula ##STR7## wherein R.sub.1 and R.sub.2 are independently
hydrogen, an amino group or a mono- or di-amino lower alkyl group;
R.sub.3 and R.sub.4 are independently hydrogen, a lower alkyl
group, an aryl group, or an acyl group with the proviso that one of
R.sub.1 and R.sub.2 must be an amino group or an mono- or diamino
lower alkyl group; and their pharmaceutically acceptable acid or
alkali addition salts; and mixtures thereof, and a carrier
therefor.
Description
BACKGROUND OF THE INVENTION
The present invention relates generally to the aging of proteins
resulting from their reaction with glucose and other reducing
sugars, and more particularly to the inhibition of the reaction of
nonenzymatically glycosylated proteins and the often resultant
formation of advanced glycosylation endproducts and cross-links.
The reaction between glucose and proteins has been known for some
time. Its earliest manifestation was ill the appearance of brown
pigments during the cooking of food, which was identified by
Maillard in 1912, who observed that glucose or other reducing
sugars react with amino acids to form adducts that undergo a series
of dehydrations and rearrangements to form stable brown pigments.
Further studies have suggested that stored and heat treated foods
undergo nonenzymatic browning as a result of the reaction between
glucose and the polypeptide chain, and that the proteins are
resultingly cross-linked and correspondingly exhibit decreased
bioavailability.
This reaction between reducing sugars and food proteins was found
to have its parallel in vivo. Thus, the nonenzymatic reaction
between glucose and the free amino groups on proteins to form a
stable, 1-deoxyketosyl adduct, known as the Amadori product, has
been shown to occur with hemoglobin, wherein a rearrangement of the
amino terminal of the beta-chain of hemoglobin by reaction with
glucose, forms the adduct known as hemoglobin A.sub.1c. The
reaction has also been found to occur with a variety of other body
proteins, such as lens crystallins, collagen and nerve proteins.
See, Bucala et al., "Advanced Glycosylation:Chemistry, Biology, and
Implications for Diabetes and Aging," in Advances in Pharmacology,
Vol. 23, pp. 1-34, Academic Press (1992).
Moreover, brown pigments with spectral and fluorescent properties
similar to those of late-stage Maillard products have also been
observed in vivo in association with several long-lived proteins,
such as lens proteins and collagen from aged individuals. An
age-related linear increase in pigment was observed in human dura
collagen between the ages of 20 to 90 years. Interestingly, the
aging of collagen can be mimicked in vitro by the cross-linking
induced by glucose; and the capture of other proteins and the
formation of adducts by collagen, also noted, is theorized to occur
by a cross-linking reaction, and is believed to account for the
observed accumulation of albumin and antibodies in kidney basement
membrane.
In Parent Application Ser. No. 798,032, now U.S. Pat. No.
4,758,583, a method and associated agents were disclosed that
served to inhibit the formation of advanced glycosylation
endproducts by reacting with an early glycosylation product that
results from the original reaction between the target protein and
glucose. Accordingly, inhibition was postulated to take place as
the reaction between the inhibitor and the early glycosylation
product appeared to interrupt the subsequent reaction of the
glycosylated protein with additional protein material to form the
cross-linked late-stage product. One of the agents identified as an
inhibitor was aminoguanidine, and the results of further testing
have borne out its efficacy in this regard.
While the success that has been achieved with aminoguanidine and
similar compounds is promising, a need continues to exist to
identify and develop additional inhibitors that broaden the
availability and perhaps the scope of this potential activity and
its diagnostic and therapeutic utility.
SUMMARY OF THE INVENTION
In accordance with the present invention, a method and compositions
are disclosed for the inhibition of the advanced glycosylation of
proteins (protein aging). In particular, the compositions comprise
agents for inhibiting nonenzymatic cross-linking (protein aging)
due to the formation of advanced glycosylation endproducts. The
agents may be selected from those materials capable of reacting
with an early glycosylation product from the reaction of glucose
with proteins and preventing further reactions. Cross-linking
caused by other reactive sugars present in vivo or in foodstuffs,
including ribose, galactose and fructose would also be prevented by
the methods and compositions of the present invention.
The agents comprise compounds having the following structural
formula: ##STR1## wherein R.sub.1 and R.sub.2 are independently
hydrogen, an amino group or a mono- or di-amino lower alkyl group;
R.sub.3 and R.sub.4 are independently hydrogen, a lower alkyl
group, an aryl group; or an acyl group with the proviso one of
R.sub.1 and R.sub.2 must be an amino group or an mono- or diamino
lower alkyl group; and their biologically or pharmaceutically
acceptable acid or alkali addition salts; and mixtures thereof, and
a carrier therefor.
The compounds utilized in the compositions of this invention appear
to react with an early glycosylation product thereby preventing the
same from later forming the advanced glycosylation end products
which lead to protein cross-links, and thereby, to protein
aging.
The present invention also relates to a method for inhibiting
protein aging by contacting the initially glycosylated protein at
the stage of the early glycosylation product with a quantity of one
or more of the agents of the present invention, or a composition
containing the same. In the instance where the present method has
industrial application, one or more of the agents may be applied to
the proteins in question, either by introduction into a mixture of
the same in the instance of a protein extract, or by application or
introduction into foodstuffs containing the protein or proteins,
all to prevent premature aging and spoilage of the particular
foodstuffs.
The ability to inhibit the formation of advanced glycosylation
endproducts carries with it significant implications in all
applications where protein aging is a serious detriment. Thus, in
the area of food technology, the retardation of food spoilage would
confer an obvious economic and social benefit by making certain
foods of marginal stability less perishable and therefore more
available for consumers. Spoilage would be reduced as would the
expense of inspection, removal, and replacement, and the extended
availability of the foods could aid in stabilizing their price in
the marketplace. Similarly, in other industrial applications where
the perishability of proteins is a problem, the admixture of the
agents of the present invention in compositions containing such
proteins would facilitate the extended useful life of the same.
Presently used food preservatives and discoloration preventatives
such as sulfur dioxide, known to cause toxicity including allergy
and asthma in animals, can be replaced with compounds such as those
described herein.
The present method has particular therapeutic application as the
Maillard process acutely affects several of the significant protein
masses in the body, among them collagen, elastin, lens proteins,
and the kidney glomerular basement membranes. These proteins
deteriorate both with age (hence the application of the term
"protein aging") and as a consequence of diabetes. Accordingly, the
ability to either retard or substantially inhibit the formation of
advanced glycosylation endproducts carries the promise of treatment
for diabetes and, of course, improving the quality and, perhaps,
duration of animal life.
The present agents are also useful in the area of personal
appearance and hygiene, as they prevent the staining of teeth by
cationic anti-microbial agents with anti-plaque properties, such as
chlorhexidine.
Accordingly, it is a principal object of the present invention to
provide a method for inhibiting the extensive cross-linking of
proteins that occurs as an ultimate consequence of the reaction of
the proteins with glucose and other reactive sugars, by
correspondingly inhibiting the formation of advanced glycosylation
endproducts.
It is a further object of the present invention to provide a method
as aforesaid which is characterized by a reaction with an initially
glycosylated protein identified as an early glycosylation
product.
It is a further object of the present invention to provide a method
as aforesaid which prevents the rearrangement and cross-linking of
the said early glycosylation products to form the said advanced
glycosylation endproducts.
It is a yet further object of the present invention to provide
agents capable of participating in the reaction with the said early
glycosylation products in the method as aforesaid.
It is a still further object of the present invention to provide
therapeutic methods of treating the adverse consequences of protein
aging by resort to the aforesaid method and agents.
It is a still further object of the present invention to provide a
method of inhibiting the discoloration of teeth by resort to the
aforesaid method and agents.
It is a still further object of the present invention to provide
compositions including pharmaceutical compositions, all
incorporating the agents of the present invention.
Other objects and advantages Will become apparent to those skilled
in the art from a consideration of the ensuing description.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
In accordance with the present invention, agents, compositions
including pharmaceutical compositions containing said agents and
associated methods have been developed which are believed to
inhibit the formation of advanced glycosylation endproducts in a
number of target proteins existing in both animals and plant
material. In particular, the invention relates to a composition
which may contain one or more agents comprising compounds having
the structural formula ##STR2## wherein R.sub.1 and R.sub.2 are
independently hydrogen, an amino group or a mono- or di-amino lower
alkyl group; R.sub.3 and R.sub.4 are independently hydrogen, a
lower alkyl group, an aryl group; or an acyl group with the proviso
one of R.sub.1 and R.sub.2 must be an amino group or an mono- or
diamino lower alkyl group; and their biologically or
pharmaceutically acceptable acid or alkali addition salts; and
mixtures thereof, and a carrier therefor.
The lower alkyl groups referred to herein contain 1-6 carbon atoms
and include methyl, ethyl, propyl, butyl, pentyl, hexyl, and the
corresponding branched-chain isomers thereof. The mono-or di-amino
alkyl groups are lower alkyl groups substituted in the chain by one
or two amino groups.
The aryl groups referred to herein encompass those containing 6-10
carbon atoms, such as phenyl and lower alkyl substituted-phenyl,
e.g., tolyl and xylyl, and phenyl substituted by 1-2 halo, hydroxy
and lower alkoxy groups. The acyl groups referred to herein are
residues of lower alkyl, aryl and heteroaryl carboxylic acids
containing 2-10 carbon atoms. They are typified by acetyl,
propionyl, butanoyl, valeryl, hexanoyl and the corresponding higher
chain and branched chain analogs thereof. The acyl radicals may
also contain one or more double bonds and/or an additional acid
functional group, e.g., glutaryl or succinyl. The heteroaryl groups
referred to above encompass aromatic heterocyclic groups containing
3-6 carbon atoms and one or more heteroatoms such as oxygen,
nitrogen or sulfur.
The halo atoms in the above formula may be fluoro, chloro, bromo
and iodo. The lower alkoxy groups contain 1-6, and preferably 1-3,
carbon atoms and are illustrated by methoxy, ethoxy, propoxy,
isopropoxy and the like. The term biologically or pharmaceutically
acceptable salts refers to salts which are tolerated by the
mammalian body and are exemplified by acid addition salts derived
from a variety of organic and inorganic acids such as sulfuric,
phosphoric, hydrochloric hydrobromic, hydroiodic, sulfamic, citric,
lactic, maleic, succinic, tartaric, cinnamic, acetic, benzoic,
glucomic, ascorbic and related acids.
Of the compounds encompassed by Formula I, certain combinations of
substituents are preferred. For instance, when R.sub.1 and R.sub.2
are both amino groups, then R.sub.3 and R.sub.4 are preferably both
hydrogen atoms. When R.sub.1 or R.sub.2 is amino group and one of
R.sub.3 or R.sub.4 is an aryl group, the other of R.sub.3 and
R.sub.4 is preferably hydrogen.
Representative compounds of the present invention are:
1,2-diamino-4-phenyl[1H]imidazole;
2-aminoimidazole sulfate;
1,2-diaminoimidazole;
1-(2,3-diaminopropyl)imidazole trihydrochloride;
4,5-diphenylimidazole-1,2-diamine;
4-(4-bromophenyl)imidazole-1,2-diamine;
4-(4-chlorophenyl)imidazole-1,2-diamine;
4-(4-hexylphenyl)imidazole-1,2-diamine;
4-(4-methoxyphenyl)imidazole-1,2-diamine;
4-phenyl-5-propylimidazole-1,2-diamine;
1-aminoimidazole;
1-amino-4-methylimidazole;
1-amino-4-(2-methylphenyl)imidazole;
2-aminoimidazole;
4-(2,4-dichlorophenyl)-2-aminoimidazole;
4-(4-methoxyphenyl)-2-aminoimidazole;
4-(4-ethoxyphenyl)-2-aminoimidazole;
4-(4-fluorophenyl)-2-aminoimidazole;
4-(4-bromophenyl)-2-aminoimidazole;
4-butyl-2-aminoimidazole;
4-ethyl-2-aminoimidazole;
4-methyl-2-aminoimidazole;
4-phenyl-2-aminoimidazole;
4-propyl-2-aminoimidazole;
1-(3-aminopropyl)imidazole;
1-(3-amlno-1-methylpropyl)imidazole;
1-(3-aminopropyl)4-5-dimethylimidazole;
1-(3-aminopropyl)4-methylimidazole;
1-(3-aminopropyl)4-phenylimidazole;
1-(3-aminopropyl)5-methyl-4-phenylimidazole;
1-(2-aminoethyl)imidazole;
2-(aminomethyl)imidazole;
2-(aminomethyl)-4-methylimidazole;
2-(3-aminopropyl)imidazole;
2-(3-aminopropyl)-4-5-dimethylimidazole;
2-(3-aminopropyl)-4-methylimidazole;
1,2 -diamino-4-methylimidazole;
1,2-diamino-4,5-dimethylimidazole; and
1,2-diamino-4-methyl-5-acetylimidazole.
The above compounds are capable of inhibiting the formation of
advanced glycosylation endproducts on target proteins. The
cross-linking of the protein to form the advanced glycosylation
endproduct contributes to the entrapment of other proteins and
results in the development in vivo of conditions such as reduced
elasticity and wrinkling of the skin, certain kidney diseases,
atherosclerosis, osteoarthritis and the like. Similarly, plant
material that undergoes nonenzymatic browning deteriorates and, in
the case of foodstuffs, become spoiled or toughened and,
consequently, inedible. Thus, the compounds employed in accordance
with this invention inhibit this late-stage Maillard effect and
intervene in the deleterious changes described above.
The rationale of the present invention is to use agents which block
the post-glycosylation step, i.e., the formation of fluorescent
chromophores, the presence of which chromophores is associated
with, and leads to adverse sequelae of diabetes and aging. An ideal
agent would prevent the formation of the chromophore and its
associate cross-links of proteins to proteins and trapping of
proteins on the other proteins, such as occurs in arteries and in
the kidney.
The chemical nature of the early glycosylation products with which
the compounds of the present invention are believed to react may
vary, and accordingly the term "early glycosylation product(s)" as
used herein is intended to include any and all such variations
within its scope. For example, early glycosylation products with
carbonyl moieties that are involved in the formation of advanced
glycosylation endproducts, and that may be blocked by reaction with
the compounds of the present invention, have been postulated. In
one embodiment, it is envisioned that the early glycosylation
product may comprise the reactive carbonyl moieties of Amadori
products or their further condensation, dehydration and/or
rearrangement products, which may condense to form advanced
glycosylation endproducts. In another scenario, reactive carbonyl
compounds, containing one or more carbonyl moieties (such as
glycolaldehyde, glyceraldehyde or 3-deoxyglucosone) may form from
the cleavage of Amadori or other early glycosylation endproducts,
and by subsequent reactions with an amine or Amadori product, may
form carbonyl containing advanced glycosylation products such as
alkylformyl-glycosylpyrroles. Several investigators have studied
the mechanism of advanced glycosylation product formation. In vitro
studies by Eble et al., (1983), "Nonenzymatic Glucosylation and
Glucose-dependent Cross-linking of Protein", J. Biol. Chem.,
258:9406-9412, concerned the cross-linking of glycosylated protein
with nonglycosylated protein in the absence of glucose. Eble et al.
sought to elucidate the mechanism of the Maillard reaction and
accordingly conducted controlled initial glycosylation of RNAase as
a model system, which was then examined under varying conditions.
In one aspect, the glycosylated protein material was isolated and
placed in a glucose-free environment and thereby observed to
determine the extent of cross-linking.
Eble et al. thereby observed that cross-linking continued to occur
not only with the glycosylated protein but with non-glycosylated
proteins as well. One of the observations noted by Eble et al. was
that the reaction between glycosylated protein and the protein
material appeared to occur at the location on the protein chain of
the amino acid lysine. Confirmatory experimentation conducted by
Eble et al. in this connection demonstrated that free lysine would
compete with the lysine on RNAase for the binding of glycosylated
protein. Thus, it might be inferred from these data that lysine may
serve as an inhibitor of advanced glycosylation; however, this
conclusion and the underlying observations leading to it should be
taken in the relatively limited context of the model system
prepared and examined by Eble et al. Clearly, Eble et al. does not
appreciate, nor is there a suggestion therein, of the discoveries
that underlie the present invention, with respect to the inhibition
of advanced glycosylation of proteins both in vitro and in
vivo.
The experiments of Eble et al. do not suggest the reactive cleavage
product mechanism or any other mechanism in the in vivo formation
of advanced glycosylation endproducts in which glucose is always
present. In fact, other investigators support this mechanism to
explain the formation of advanced glycosylated endproducts in vivo
(see for example Hayase et al, J. Biol. Chem., 263, pp. 3758-3764
(1989); Sell and Monnier, J. Biol. Chem., 264, pp. 21597-21602
(1989); Oimomi et al., Agric. Biol. Chem., 5.3(6):1727-1728 (1989);
and Diabetes Research and Clinical Practice, 6:311-313 (1989).
Accordingly, the use of lysine as an inhibitor in the Eble et al.
model system has no bearing upon the utility of the compounds of
the present invention in the inhibition of advanced glycosylated
endproducts formation in the presence of glucose in vivo, and the
amelioration of complications of diabetes and aging.
The compositions useful in the present invention comprise or
contain agents capable of reacting with the active carbonyl
intermediate of an early glycosylation product. Suitable agents are
the compounds of Formula I of the present invention.
The present invention likewise relates to methods for inhibiting
the formation of advanced glycosylation endproducts, which comprise
contacting the target proteins with a composition of the present
invention. In the instance where the target proteins are contained
in foodstuffs, whether of plant or animal origin, these foodstuffs
could have applied to them by various conventional means a
composition containing the present agents.
In the food industry, sulfites were found years ago to inhibit the
Maillard reaction and are commonly used in processed and stored
foods. Recently, however, sulfites in food have been implicated in
severe and even fatal reactions in asthmatics- As a consequence,
the sulfite treatment of fresh fruits and vegetables has been
banned. The mechanism for the allergic reaction is not known.
Accordingly, the present compositions and agents offer a nontoxic
alternative to sulfites in the treatment of foods in this
manner.
As is apparent from a discussion of the environment of the present
invention, the present methods and compositions hold the promise
for arresting the aging of key proteins both in animals and plants,
and concomitantly, conferring both economic and medical benefits as
a result thereof. In the instance of foodstuffs, the administration
of the present composition holds the promise for retarding food
spoilage thereby making foodstuffs of increased shelf life and
greater availability to consumers. Replacement of currently-used
preservatives, such as sulfur dioxide known to cause allergies and
asthma in humans, with non-toxic, biocompatible compounds is a
further advantage of the present invention.
The therapeutic implications of the present invention relate to the
arrest of the aging process which has, as indicated earlier, been
identified in the aging of key proteins by advanced glycosylation
and cross-linking. Thus, body proteins, and particularly structural
body proteins, such as collagen, elastin, lens proteins, nerve
proteins, kidney glomerular basement membranes and other
extravascular matrix components would all benefit in their
longevity and operation from the practice of the present invention.
The present invention thus reduces the incidence of pathologies
involving the entrapment of proteins by cross-linked target
proteins, such as retinopathy, cataracts, diabetic kidney disease,
glomerulosclerosis, peripheral vascular disease, arteriosclerosis
obliterans, peripheral neuropathy, stroke, hypertension,
atherosclerosis, osteoarthritis, periarticular rigidity, loss of
elasticity and wrinkling of skin, stiffening of joints,
glomerulonephritis, etc. Likewise, all of these conditions are in
evidence in patients afflicted with diabetes mellitus. Thus, the
present therapeutic method is relevant to treatment of the noted
conditions in patients either of advanced age or those suffering
from one of the mentioned pathologies.
Protein cross-linking through advanced glycosylation product
formation can decrease solubility of structural proteins such as
collagen in vessel walls and can also trap serum proteins, such as
lipoproteins to the collagen. Also, this may result in increased
permeability of the endothelium and consequently covalent trapping
of extravasated plasma proteins in subendothelial matrix, and
reduction in susceptibility of both plasma and matrix proteins to
physiologic degradation by enzymes. For these reasons, the
progressive occlusion of diabetic vessels induced by chronic
hyperglycemia has been hypothesized to result from excessive
formation of glucose-derived cross-links. such diabetic
macrovascular changes and microvascular occlusion can be
effectively prevented by chemical inhibition of advanced
glycosylation product formation utilizing a composition and the
methods of the present invention.
Studies indicate that the development of chronic diabetic damage in
target organs is primarily linked to hyperglycemia so that tight
metabolic control would delay or even prevent end-organ damage. See
Nicholls et al., Lab. Invest., 60, No. 4, p. 486 (1989), which
discusses the effects of islet isografting and aminoguanidine in
murine diabetic nephropathy. These studies further evidence that
aminoguanidine diminishes aortic wall protein cross-linking in
diabetic rats and confirm earlier studies by Brownlee et al.,
Science, 232, pp. 1629-1632 (1986) to this additional target organ
of complication of diabetes. Also, an additional study showed the
reduction of immunoglobulin trapping in the kidney by
aminoguanidine (Brownlee et al., Diabetes, 35, Suppl. 1, p. 42A
(1986)).
Further evidence in the streptozotocin-diabetic rat model that
aminoguanidine administration intervenes in the development of
diabetic nephropathy was presented by Brownlee et al., 1988, supra,
with regard to morphologic changes in the kidney which are
hallmarks of diabetic renal disease. These investigators reported
that the increased glomerular basement membrane thickness, a major
structural abnormality characteristic of diabetic renal disease,
was prevented with aminoguanidine.
Taken together, these data strongly suggest that inhibition of the
formation of advanced glycosylation endproducts (AGEs), by the
teaching of the present invention, may prevent late, as well as
early, structural lesions due to diabetes, as well as changes
during aging caused by the formation of AGEs.
Diabetes-induced changes in the deformability of red blood cells,
leading to more rigid cell membranes, is another manifestation of
cross-linking and aminoguanidine has been shown to prevent it in
vivo. In such studies, New Zealand White rabbits, with induced,
long-term diabetes are used to study the effects of a test compound
on red blood cell (RBC) deformability (dr). The test compound is
administered at a rate of 100 mg/kg by oral garage to diabetic
rabbits.
A further consequence of diabetes is the hyperglycemiainduced
matrix bone differentiation resulting in decreased bone formation
usually associated with chronic diabetes. In animal models,
diabetes reduces matrixinduced bone differentiation by 70%.
In the instance where the compositions of the present invention are
utilized for in vivo or therapeutic purposes, it may be noted that
the compounds or agents used therein are biocompatible.
Pharmaceutical compositions may be prepared with a therapeutically
effective quantity of the agents or compounds of the present
invention and may include a pharmaceutically acceptable carrier,
selected from known materials utilized for this purpose. Such
compositions may be prepared in a variety of forms, depending on
the method of administration. Also, various pharmaceutically
acceptable addition salts of the compounds of Formula I may be
utilized.
A liquid form would be utilized in the instance where
administration is by intravenous, intramuscular or intraperitoneal
injection. When appropriate, solid dosage forms such as tablets,
capsules, or liquid dosage formulations such as solutions and
suspensions, etc., may be prepared for oral administration. For
topical or dermal application to the skin or eye, a solution, a
lotion or ointment may be formulated with the agent in a suitable
vehicle such as water, ethanol, propylene glycol, perhaps including
a carrier to aid in penetration into the skin or eye. For example,
a topical preparation could include up to about 10% of the compound
of Formula I. Other suitable forms for administration to other body
tissues are also contemplated.
In the instance where the present method has therapeutic
application, the animal host intended for treatment may have
administered to it a quantity of one or more of the agents, in a
suitable pharmaceutical form. Administration may be accomplished by
known techniques, such as oral, topical and parenteral techniques
such as intradermal, subcutaneous, intravenous or intraperitoneal
injection, as well as by other conventional means. Administration
of the agents may take place over an extended period of time at a
dosage level of, for example, up to about 30 mg/kg.
As noted earlier, the invention also extends to a method of
inhibiting the discoloration of teeth resulting from nonenzymatic
browning in the oral cavity which comprises administration to a
subject in need of such therapy an amount effective to inhibit the
formation of advanced glycosylation endproducts of a composition
comprising an agent of structural Formula I.
The nonenzymatic browning reaction which occurs in the oral cavity
results in the discoloration of teeth. Presently used anti-plaque
agents accelerate this nonenzymatic browning reaction and further
the staining of the teeth. Recently, a class of cationic
antimicrobial agents with remarkable anti-plaque properties have
been formulated in oral rinses for regular use to kill bacteria in
the mouth. These agents, the cationic antiseptics, include such
agents as alexidine, cetyl pyridinium chloride, chlorhexidine
gluconate, hexetidine, and benzalkonium chloride.
Tooth staining by chlorhexidine and other anti-plaque agents
apparently results from the enhancement of the Maillard reaction.
Nordbo, J. Dent. Res., 58, p. 1429 (1979) reported that
chlorhexidine and benzalkonium chloride catalyze browning reactions
in vitro. Chlorhexidine added to mixtures containing a sugar
derivative and a source of amino groups underwent increased color
formation, attributed to the Maillard reaction. It is also known
that use of chlorhexidine results in an increased dental pellicle.
Nordbo proposed that chlorhexidine resulted in tooth staining in
two ways: first, by increasing formation of pellicle which contains
more amino groups, and secondly, by catalysis of the Maillard
reaction leading to colored products.
In accordance with this method, the compounds of Formula I are
formulated into compositions adapted for use in the oral cavity.
Particularly suitable formulations are oral rinses and toothpastes
incorporating the active agent.
In the practice of this invention, conventional formulating
techniques are utilized with nontoxic, pharmaceutically acceptable
carriers typically utilized in the amounts and combinations that
are well-known for the formulation of such oral rinses and
toothpastes.
The agent of Formula I is formulated in compositions in an amount
effective to inhibit the formation of advanced glycosylation
endproducts. This amount will, of course, vary with the particular
agent being utilized and the particular dosage form, but typically
is in the range of 0.01% to 1.0%, by weight, of the particular
formulation.
Additionally, since the agents of the aforesaid method are
concentrated in the salivary glands upon oral ingestion or
parenteral administration, they can be so administered. This
concentration in the salivary glands results in their secretion
into saliva, the net result being that they are functionally placed
in the oral cavity where they can effect their desired method. For
such administration, the particular agent can be formulated in any
conventional oral or parenteral dosage form. A particularly
desirable dosage form is the incorporation of the agent into a
vitamin tablet or fluoride tablet so as to maximize patient, and
particularly juvenile patient, compliance.
The known compounds encompassed by Formula I are conveniently
prepared by chemical syntheses well-known in the art. Certain of
the compounds encompassed by Formula I are well-known compounds
readily available from chemical supply houses and/or preparable by
synthetic methods specifically published therefor.
A preferred process for the preparation of the novel compound of
formula I wherein R.sub.2 is an amino group and R.sub.1, R.sub.3
and R.sub.4 are all hydrogen involves the reaction of 5-methyl
isothiosemicarbazide tosylate and aminoacetaldehyde dimethyl acetal
in a polar solvent under reflux conditions. Alternately, the
compound can be prepared by reaction of hydroxylamine-O-sulfonic
acid with 2-aminoimidazole under basic conditions.
Compounds described in the chemical and patent literature or
directly preparable by methods described therein and encompassed by
Formula I are those such as 1,2-diamino-4-phenyl-[1H]imidazole (See
Chem. Ber., 101, 315-3162 (1968); 2-aminoimidazole sulfate;
1,2-diaminoimidazole; and
1-(2,3-diaminopropyl)imidazole trihydrochloride; and their
biologically or pharmaceutically acceptable acid or alkali addition
salts.
EXAMPLE I
The following method was used to evaluate the ability of the
compounds of the present invention to inhibit glucose-mediated
development of fluorescence of bovine serum albumin (BSA), a
measure of cross-linking. Compounds were incubated under aseptic
conditions at a concentration of 1 mM with 400 mM glucose and 100
mg/mL BSA in a 1.5 M sodium phosphate buffer, pH 7.4.
Samples of the incubation mixture were taken immediately and after
1 week incubation at 37.degree. C. for measurement of fluorescence.
For each test compound, control incubations in buffer were made of
compound alone (C), compound plus glucose (G+C), and compound plus
BSA (B+C). An additional set of incubations of glucose and BSA
(B+G) were prepared as the baseline controls against which were
measured the ability of the compounds to inhibit. Each incubation
was made in triplicate.
Fluorescence (excitation, 370 nm; emission, 440 nm) was measured on
each sample after a 100-fold dilution in distilled water.
The % inhibition of browning of each test compound was calculated
as follows. Each .DELTA.F represents the fluorescence measurement
of that sample after 1 week incubation less its fluorescence before
incubation. ##EQU1## where B=BSA, G=glucose, and C=test compound.
Percent inhibition of browning by various test compounds at 1
mM:
______________________________________ 0% no inhibitor; 33.5%
1,2-diamino-4-phenyl-[1H]imidazole; 63.3% 2-aminoimidazole sulfate;
and 51.5% 1,2-diaminoimidazole.
______________________________________
The above experiments suggest that this type of drug therapy may
have benefit in reducing the pathology associated with the advanced
glycosylation of proteins and the formation of cross-links between
proteins and other macromolecules. Drug therapy may be used to
prevent the increased trapping and cross-linking of proteins that
occurs in diabetes and aging which leads to sequelae such as
retinal damage, and extra-vascularly, damage to tendons, ligaments
and other joints. This therapy might retard atherosclerosis and
connective tissue changes that occur with diabetes and aging. Both
topical, oral, and parenteral routes of administration to provide
therapy locally and systemically are contemplated.
EXAMPLE 2
______________________________________ Tablet mg/tablet
______________________________________ Compound of Formula I 50
Starch 50 Mannitol 75 Magnesium stearate 2 Stearic acid 5
______________________________________
The compound, a portion of the starch and the lactose are combined
and wet granulated with starch paste. The wet granulation is placed
on trays and allowed to dry overnight at a temperature of
45.degree. C. The dried granulation is comminuted in a comminutor
to a particle size of approximately 20 mesh. Magnesium stearate,
stearic acid and the balance of the starch are added and the entire
mix blended prior to compression on a suitable tablet press. The
tablets are compressed at a weight of 232 mg. using a 11/32" punch
with a hardness of 4 kg. These tablets will disintegrate within a
half hour according to the method described in USP XVI.
EXAMPLE 3
______________________________________ Lotion mg/g
______________________________________ Compound of Formula I 1.0
Ethyl alcohol 400.0 Polyethylene glycol 400 300.0 Hydroxypropyl
cellulose 5.0 Propylene glycol to make 1.0 g
______________________________________
EXAMPLE 4
______________________________________ Oral Rinse
______________________________________ Compound of Formula I: 1.4%
Chlorhexidine gluconate 0.12% Ethanol 11.6% Sodium saccharin 0.15%
FD&C Blue No. 1 0.001% Peppermint Oil 0.5% Glycerine 10.0%
Tween 60 0.3% Water to 100%
______________________________________
EXAMPLE 5
______________________________________ Toothpaste
______________________________________ Compound of Formula I: 5.5%
Sorbitol, 70% in water 25% Sodium saccharin 0.15% Sodium lauryl
sulfate 1.75% Carbopol 934, 6% dispersion in water 15% Oil of
Spearmint 1.0% Sodium hydroxide, 50% in water 0.76% Dibasic calcium
phosphate dihydrate 45% Water to 100%
______________________________________
EXAMPLE 6
To further study the ability of inhibitors of nonenzymatic browning
to prevent the discoloration of protein on a surface, such as that
which occurs on the tooth surface, the following surface browning
experiment is performed. As a substitute for a pellicle-covered
tooth surface, unexposed and developed photographic paper is used
to provide a fixed protein (gelatin, i.e., collagen) surface on a
paper backing. Five millimeter circles are punched and immersed for
one week at 50.degree. C. in a solution of 100 mM
glucose-6-phosphate in a 0.5 M phosphate buffer, pH 7.4, containing
3 mM sodium azide. Glucose-6-phosphate is a sugar capable of
participating in nonenzymatic browning at a more rapid rate than
glucose. In addition to the glucose-6-phosphate, chlorhexidine
and/or a compound of Formula I are included. After incubation, the
gelatin/paper disks are rinsed with water, observed for brown
color, and photographed.
Incubation of the disks in glucose-6-phosphate alone shows slight
brown color versus disks soaked in buffer alone. Inclusion of
chlorhexidine (in the form of Peridex.RTM. at a final concentration
of 0.04% chlorhexidine) shows significant browning. Addition of a
compound of Formula I to the chlorhexidine completely inhibits
browning of the gelatin, as does inclusion of a compound of Formula
I in the absence of chlorhexidine.
The slight brown color formed by the action of glucose-6-phosphate
on the gelatin surface alone and its prevention by a compound of
Formula I demonstrates the utility of the present invention in
preventing nonenzymatic browning of tooth surfaces. The enhanced
browning in the presence of chlorhexidine and its prevention with a
compound of Formula I demonstrates the utility of the present
invention in preventing the anti-plaque agent-enhanced nonenzymatic
browning which occurs with chlorhexidine.
EXAMPLE 7
Preparation of 1,2-Diaminoimidazole
A mixture of S-methyl-isothiosemicarbazide tosylate (2.77 g, 10
mmol) and aminoacetaldehyde dimethyl acetal (1.26 g, 12 mmol) was
taken up in ethyl alcohol (10 ml) and refluxed overnight. It was
cooled at room temperature and p-toluenesulfonic acid (1.72 g, 10
mmol) was added. The reaction mixture was then again refluxed
overnight. The solvent was evaporated and the residue was
crystallized from ethyl alcohol to yield 1.518 (56%) of the title
compound, mp-180.degree.-181.degree. C.
This invention may be embodied in other forms or carried out in
other ways without departing from the spirit or essential
characteristics thereof. The present disclosure is therefore to be
considered as in all respects illustrative and not restrictive, the
scope of the invention being indicated by the appended claims, and
all changes which come within the meaning and range of equivalency
are intended to be embraced therein.
* * * * *